Interest in mutually exclusive pairs of bioorthogonal labeling reagents continues to drive the design of new compounds that are capable of fast and predictable reactions. The ability to easily modify S-, N-, and O-containing cyclooctynes (SNO-OCTs) enables electronic tuning of various SNO-OCTs to influence their cycloaddition rates with Type I-III dipoles. As opposed to optimizations based on just one specific dipole class, the electrophilicity of the alkynes in SNO-OCTs can be manipulated to achieve divergent reactivities and furnish mutually orthogonal dual ligation systems. Significant reaction rate enhancements of a difluorinated SNO-OCT derivative, as compared to the parent scaffold, were noted, with the second-order rate constant in cycloadditions with diazoacetamides exceeding 5.13 M-1 s-1. Computational and experimental studies were employed to inform the design of triple ligation systems that encompass three orthogonal reactivities. Finally, polar SNO-OCTs are rapidly internalized by mammalian cells and remain functional in the cytosol for live-cell labeling, highlighting their potential for diverse in vitro and in vivo applications.